Triton x

Cerebral organoids were fixed in 4% paraformaldehyde and incubated with sucrose gradient solutions (10, 20 and 30% in PBS) for 15 min each. They were then embedded in optimal cutting temperature compound (OCT) and frozen in liquid nitrogen. Twenty-micron-thick sections were permeabilized with 0.3% Triton-X (Vetec Química Fina Ltda, Rio de Janeiro, Rio de Janeiro, BR) for 5 min at room temperature. Subsequently, the tissue sections were blocked with 3% bovine serum albumin (BSA, Sigma) and 5% normal goat serum (NGS, Invitrogen) in PBS (block solution) for 1 h and incubated overnight at 4 °C with the specified primary antibody diluted in blocking solution. The primary antibodies used were mouse anti-Nestin (1:200; Merck), rabbit anti-BLBP (1:200; Millipore), and rabbit anti-FOXG1 (1:00; Santa Cruz Biotechnology, Inc.). Secondary antibodies were Alexa Fluor 546 goat anti-mouse (1:1,000; Molecular Probes) and Alexa Fluor 488 goat anti-rabbit (1:400; Molecular Probes). Negative controls were obtained by omitting the primary antibodies; in all cases, no reactivity was observed. DAPI (4′,6-diamidino-2-phenylindole, 1 mg/mL, Sigma) was used to stain nuclei. Images were acquired using an Operetta Imaging System (Perkin Elmer Inc.).